Method for producing a polyphenolic composition from barley malt

ABSTRACT

Method for the production of a polyphenolic composition from barley malt, including the fundamental steps of: grinding of the malt grains and splitting into two portions, 20% and 80%; mixing each of the two portions with water to obtain an A mixture and a B mixture, the A mixture being prepared with the 20% portion to obtain a mixture of the malt in water at a final concentration between 9.5% and 20%, the B mixture being prepared with the 80% portion to obtain a mixture of the malt in water at a final concentration between 33% and 60%; thermal cycle; separation of the liquid component from the solid component; boiling of the liquid component and the addition of hops; rapid cooling of the wort; storage; in which said thermal cycle consists of a first phase and a second phase, in which the first phase applies to the 20% portion (Mixture A) and the second phase during which Mixture B is added to Mixture A. The relative polyphenolic composition obtained according to this method is characterized by the fact that it can be in liquid, powder, dry, and lyophilized form, its use and relative beverage or beer according to the present method not obtained with fermentation methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 16/641,888, filed Feb. 25, 2020, which is a 371 National StageApplication of International Patent Application Serial No.PCT/1B2018/056283, filed Aug. 20, 2018, which claims the benefit ofItalian Patent Application No. 102017000096298, filed Aug. 25, 2017, theentire contents of which are incorporated herein by reference.

BACKGROUND

This invention refers in general to the food, nutraceutical, andpharmaceutical sectors applied to cereal by-products, more specificallyto a polyphenolic composition obtained from cereals. In detail, thisinvention refers to a method for producing a polyphenolic compositionfrom malt and hops; the abovementioned polyphenolic composition hasevident healthy effects. More specifically, this polyphenoliccomposition obtained from malt and hops is characterized by a highcontent of natural polyphenols, demonstrating significant anti-oxidantand anti-tumor effects.

The healthy effects of some alcoholic beverages like wine and beer areknown, if consumed in moderate amounts; some studies have demonstratedthe correlation between the consumption of beer and the prevention ofheart diseases, cancer, and osteoporosis (Gerhauser 2005; Kondo 2004,Rivero et al. 2005), as well as the correlation between beer consumptionand the anti-oxidant power of the serum (Ghiselli et al. 2000).

The positive association between moderate consumption of alcoholicbeverages and low risk of degenerative diseases was also linked to thepolyphenol content.

As is known, phenolic substances are a class of natural organiccompounds characterized by the presence of at least one aromatic ring,mono- or multi-substituted by hydroxyl groups. Among these, flavonoidshave a base structure with two aromatic rings linked by a heterocycliccompound through either a hydroxylated, glycosylated, or methylatedconnection. The most common vegetable flavonoids are the flavans, whichare more commonly present in beer, (+) catechin, (−) epicatechin,gallocatechin, and epigallocatechin, in a monomer, dimer, trimer, orpolymer form.

Phenolic compounds as donors of hydrogen possess an anti-oxidant qualitylinked in hydroxyl groups linked to aromatic structures and to thegeometry of the molecule; also the degree of polymerization influencesthe anti-oxidant action.

In particular, it was seen that the anti-oxidant action of beer iscorrelated to the total polyphenol content (Rivero et al., 2005; Tedescoet al., 2005) and that beers normally have a total polyphenol content ofapproximately 489 mg/l, or double that of a light nonalcoholic beer. Itis also known that about 20-30% of the polyphenols in beer derive fromhops, while 70-80% derive from malt (Benitez et al., 1997).

Indeed, recent studies have focused on the interesting biologicalproperties of compounds deriving from hops and found in beer, likexanthohumol, isoxanthohumol and bitter acids. These molecules seem to beable to perform an anti-tumor action in both cellular and animal models.In particular, the xanthohumol acts as an anti-tumor agent both in vitroand in ex vivo models of chronic lymphocytic leukemia (Lust et al.,2005; Miranda et al., 1999).

Several works were known to be state-of-the-art concerning thechemo-preventive effectiveness of beer in experimental carcinogenesismodels; for example, Nelson & Salmeson (1985) demonstrated that thechronic consumption of beer reduced the formation of gastrointestinaltumors induced by dimethylthirizine in rats.

On the other hand, Tedesco et al. (2005) verified the high probabilitythat the synergic effect of phenolic compounds in beer triggers theinhibition of the proliferation of tumor cells and the induction ofapoptosis. Indeed, the anti-proliferative activity of individualmolecules added to the tumor cells, at an average concentration found incommercial beers used, was absent in tests where the biological activityof the lyophilizate in toto was measured.

With reference to the health properties of some alcoholic andnon-alcoholic beverages made with cereals, various patent documentsconcerning technical status are also known. The patent documentUS20080261897 describes a formulation containing epigallocatechin toreduce bitterness; in the patent in question, there is no reference to aspecific polyphenol composition and the method used to obtain thebeverage; lastly, there is no reference to the biological activity ofsaid composition.

Patent US2005266120 describes a method for the production of a fermentedbeverage similar to beer and non-alcoholic, fermented using a slowfermentation process; this beverage contains a very low quantity ofalcohol—0,5% (p/v)—obtained by the fermentation of fungi of the genreMonascus; these fungi ferment alcohol slowly and have a highanti-oxidant activity to control the fermentation of the alcohol.

The patent document US20080213433 describes a non-alcoholic beveragewith a polyphenol base, making particular reference to thetrans-resveratrol in a concentration between 5-300 mg/kg. This documentalso contains a description of a method that provides for the additionof this polyphenol composition to a variety of beverages, includingcoffee and other barley products.

The need to create a polyphenolic composition to add to the beveragesmarketed, or directly produced in beverage form, derives from the factthat normal cooking processes, like in the production of beer, ortoasting, like in the production of coffee, cause a depletion of thepolyphenol content naturally found in the original foodstuff. Thisinvention describes an all-natural method for preserving the highquantity of natural polyphenols present in barley malt to obtain acharacteristic polyphenol composition. Moreover, it has been noticed, inthe technical sector of reference, the need to provide a beverage thathas a high content of polyphenol naturally present in the foods, butthat is a totally non-alcoholic product (0% alcohol content); saidbeverage is different from a non-alcoholic beer in that by Italian lawthe denomination ‘non-alcoholic’ beer is reserved for products withPlato degrees no less than 3 and no higher than 8 and with an alcoholicstrength by volume no higher than 1.2%. The denomination ‘light beer’ isreserved for the product with a Plato degree no less than 5 and nohigher than 10.5 and with an alcoholic strength by volume between 1.2%and 3.5%. The denomination “beer” is reserved for the product with aPlato degree higher than 10.5 and with an alcoholic strength by volumehigher than 3.5%.

SUMMARY

In the light of the above, the scope of the present invention istherefore to provide a natural polyphenolic composition starting frommalt and hops that preserves most of the polyphenols present in the rawmaterials intact, thereby obtaining a composition characterized by highanti-oxidant and anti-tumor activities. It is also scope of the presentinvention to provide a method for producing the above-mentionedpolyphenolic composition that preserves the largest possible quantity ofpolyphenols present in the malt, thereby obtaining a compositioncharacterized by high anti-oxidant and anti-tumor activities. Saidmethod does not provide for the use of the fermentation phase.

Yet another scope of this invention is to provide a polyphenoliccomposition starting from unfermented malt and hops in the form of acompletely non-alcoholic functional beverage, containing 0% alcohol,naturally rich in polyphenols and not artificially enriched withpolyphenols.

Said beverage obtained from unfermented malt, naturally rich inpolyphenols, is completely non-alcoholic. As it is a beverage with noalcohol, 0% alcohol content, it can be used by any type of user:children, the elderly, or people with specific pathologies for which theconsumption of polyphenolic compositions is suggested but theconsumption of alcoholic beverages is not recommended, even if inmoderate quantities.

This invention therefore provides a method for the production of apolyphenolic composition starting from unfermented malt.

In a preferred embodiment, this invention provides a polyphenoliccomposition starting from unfermented malt comprising polyphenols suchas: caffeic acid, m-Coumaric acid, p-hydroxybenzoic acid, sinapinicacid, epicatechin, protocatechuic acid, catechin, p-Coumaric acid,ferulic acid, vanillic acid, chlorogenic acid and their mixtures, andcharacterized in that it contains chlorogenic acid. Other aspects ofthis invention, including the forms of realization, medical uses,anti-oxidant and anti-tumor health benefits are described in thefollowing claims attached.

The aforementioned claims are understood to have been included herein.This invention will become more evident from the detailed descriptionthat follows, with reference to the drawings attached that illustrate anembodiment of the polyphenolic composition starting from unfermentedmalt and hops and the relative production method, where:

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is the block diagram of the production method of the polyphenoliccomposition obtained from unfermented malt and hops;

FIG. 2 illustrates the effect of the polyphenolic composition thisinvention provides on the formation of total ACF (aberrant crypt foci) A(FIG. 2A) and foci constituted by 4 or more aberrant crypts (FIG. 2B)induced in the colons of mice by AOM (azomethane);

FIG. 3 illustrates the effect of the polyphenolic composition thisinvention provides on the formation of polyps (FIG. 3A) and tumors (FIG.3B) induced in the colons of mice by AOM (azomethane);

FIG. 4 shows an assay with peroxides (expressed as equivalents ofhydrogen peroxide) in samples of serum of mice treated with theconcentrations indicated of polyphenolic composition provided by thisinvention in the presence/absence of AOM; FIG. 5 is a graph of theexemplary thermal cycle of the production method of the polyphenoliccomposition that this invention provides.

It is provided, in the following, the description of an exemplaryembodiment, as such not restricted, of the production method of thepolyphenolic composition starting from unfermented malt and hops and therelative composition obtained.

DESCRIPTION OF THE INVENTION

In the following all the % indicated are % in weight.

The production method of the above-mentioned polyphenolic compositionprovides the use, by way of example, of the following raw materialsmixed with water: light and dark barley malt in varying proportionsdepending on the color desired for the final product, the larger theproportion of dark malt (more toasted) the more intense the color of thefinished product; hops; acidifiers, those usually used in this field,preferably citric acid, lactic acid, and orthophosphoric acid, usefulfor bringing the pH of the final mixture within the interval of 4.0-4.6,preferably 4.2.

With reference to FIG. 1 , the method for the production of theabove-mentioned polyphenolic composition comprising the following steps:

1. grinding of malt grains and splitting in two portions, 20% and 80%,101a;

2. mixing 101 b each of the two portions with water to obtain a mixtureA and a mixture B. The A mixture is prepared with the 20% portion toobtain a mixture of malt in water with a final concentration between9.5% and 20%. Mixture B is prepared with the 80% portion to obtain amixture of malt in water with a final concentration between 33% and 60%.An example of production requires that the A mixture be obtained with360 kg of ground malt in 28±10 hl of water, and that the B mixture beobtained with 1470 kg of ground malt in 35±10 hl of water.

3. thermal cycle 102 with two consecutively phases, as indicated below;

4. separation of the liquid component from the solid component (thespent grains) 103 at the final temperature reached in the thermal cycle(76±5° C.) and subsequent washing of the spent grains at the sametemperature (76±5° C.);

5. boiling of the liquid component resulting from step 4 and addition ofhops 104 (thereby obtaining the beer wort);

6. rapid cooling of the wort 105 at a rate of 1,85±0,8° C. per minute;

7. storage at a final temperature after cooling 106 without additionalyeasts;

8. optional purification treatment 107;

9. optional treatment with natural preservatives 108;

10. optional bottling 109;

11. optional thermal treatment to prolong preservation, actually apasteurization 110;

12. optional drying 111 with known techniques, for examplelyophilization 112.

More specifically, the production method for the polyphenoliccomposition from unfermented malt is characterized by a thermal cycle(step 3) which makes it possible to preserve the largest quantity ofpolyphenols naturally present in the malt, immediately after itsgrinding.

This thermal cycle consists of a first phase and a second phase.

The first phase of this thermal cycle is applied only to the 20% of themalt mixture (mixture A), and the second phase is applied to the entirebatch, or the 20% previously treated to which the remaining 80% (mixtureB) has been added.

The first phase of the thermal cycle is characterized by a heatingprocess when an initial gelatinization of the starch takes place. Theheating in conducted fora period of 1-2 minutes, preferably 1 minute,until it reaches a temperature of 71° C., preferably 66±5° C., or morepreferably 66° C. This heating phase is followed by a holding pause oftemperature maintenance for 20-30 minutes, preferably 20 minutes,followed by an increase in the temperature for about 10-20 minutes,preferably 15-16 minutes, until the mixture boils (typically at 100°C.).

Following the heating, a pause in the boiling is required for 10-25minutes, followed by cooling phase lasting 10-18 minutes, preferably12±2 minutes, more preferably 14 minutes, until the mixture reaches atemperature between 45° C. and 55° C.

The second phase of the thermal cycle is applied to all the mixture,which means that the remaining portion (mixture B) is added, after beingheated to the same temperature reached by mixture A, or between 45-55°C., preferably 50° C. The temperature between 45-55° C. is maintainedfor 15-30 minutes and constitutes the pause proteolytic phase duringwhich the protease enzyme breaks up the proteins into polypeptidefractions. Then mixture undergoes successive heating in three stageswith temperatures increasing between 60° C. and 80° C. The first stageof heating to 63±2° C. is reached in 7-11 minutes and maintained forabout 37-42 minutes.

The second heating stage at 72±5° C. is reached in 4-10 minutes andmaintained for about 37-42 minutes;

The third heating stage at 76±2° C. is reached in 3-10 minutes andmaintained for about 100-300 minutes.

The first two stages of heating constitute the saccharification stagesthat make it possible for the beta-amylase and alpha-amylase enzymes tobreak up the starches.

The final stage of heating is followed by the pouring of the mixtureinto a filtering vat. Later, at the same temperature as the said thermalcycle, comes the separation, for example for filtration and/ordecanting, of the dregs (essentially the spent grains) from the liquidcomponent (the wort) Once separated, the dregs are washed with water andthe washing water is added to the wort. It is preferable to carry out atleast three washes of the spent grains.

The wort, maintained at the temperature of the third heating stage, iscollected in a storage container (optional phase for the cooking roommanagement needs, and can be omitted) and transferred to a heater wherethe hops are added in a quantity of up to 0.5%, preferably 0.2%-0.3%. Byway of example, for a quantity of malt of 1830 kg, 4 kg of hops extractand 1.5 kg of spalt hops can be used (these quantities may vary inrelation to the alpha acids content). This wort with the hops added thenundergoes a further heating and boiling phase lasting between 40-100minutes, typically at 108° C. for about 60 minutes. In a normal beerproduction process, the hops are added to make the beer more bitter,thanks to the component of the alpha acids, and aromatic, thanks to thebeta acids and essential oils. Successively, steam is preferably addedto the wort at a temperature>100° C., from the bottom up to eliminateunpleasant volatile compounds. The boiling phase of the wort with thehops added is followed by a cooling until a temperature between 2-10°C., preferably 2-4° C., is reached.

After cooling, wort is sent into storage and aging tanks for at least 3days. After cooling the wort is sent to a storage tank and aged for atleast 3 days. This aging process may be prolonged for several months.This step does not call for any yeasts to be added, contrary to the beerproduction process where yeasts are added at this step. Not adding theseyeasts influences the polyphenolic composition and the obtaining of aproduct with a 0% alcohol content; it is indeed known that yeastsnormally use fermentable sugars to obtain alcohol and carbon dioxide.

Once the aging step has ended, there is another optional step, theelimination of impurities 107, this step 107 may take place through asimple decanting or by means of filtering with Diatomaceous earth toobtain the final beverage. Step 108, which is optional, will be atreatment of the beverage with natural preservatives; in a preferredembodiment, these preservatives would be coloring malt and ascorbicacid.

Other optional steps of bottling and pasteurization may follow; or evendrying and/or lyophilization with known technical procedures.

The polyphenolic composition obtained with the method provided with thisinvention is characterized by the following ratios between the mainphenolic components:

TABLE 1 Polyphenolic composition obtained with the method provided withthis invention Main phenolic compounds Ratio ranges relative to the ofthe polyphenolic phenolic compositions (Caffeic Acid compositionmolecule concentration is set as equal to 1) Caffeic Acid 1 ChlorogenicAcid 0.83-1.01 m-Coumaric Acid 1.04-1.27 P-hydroxybenzoic Acid 1.13-1.39Sinapinic acid 1.33-1.63 Epicatechin 1.67-2.04 Protocatechuic Acid1.73-2.11 Catechin 2.39-2.93 p-Coumaric Acid 5.13-6.27 Ferulic Acid8.46-10.3 Vanillic acid 2.47-3.01

The method provided by this invention differs from standard methods ofbeer production for the fact that, while in a beer production processthe polyphenolic content tends to be reduced due to the cloudiness andthe flavor granted by some polyphenols, in the method provided in theinvention each passage was modified in order to preserve thepolyphenolic content of the malt, thereby obtaining an originalpolyphenolic composition that, in its liquid form, is different fromthose commonly known to be contained in beers. The abovementioned methoddoes not need yeasts to be added and therefore fermentation is notinduced, making it possible to obtain a beverage from malt and hops thatcontains 0% alcohol and preserves a high polyphenol content, as well asobtain the polyphenolic composition as illustrated in Table 1. Thepolyphenolic composition from unfermented malt and hops that thisinvention provides is characterised by the fact that it can be in eitherliquid or powdered form (dried or lyophilized). Said composition can beused as a food or beverage, as a functional food, as dietary supplement,or as a pharmaceutical in typical pharmaceutical forms like, by way ofexample and not limited to:

solid pharmaceutical forms (tablets, pills, hard capsules, powders,granules, suppositories)

semi-solid pharmaceutical forms (gels, ointments, lubricants, pastes)

liquid pharmaceutical forms (syrups, vials, drops, eye drops).

Another embodiment of the polyphenolic composition from unfermented maltand hops provided by this invention is related to its use in a genericnon-alcoholic beverage that, by way of example but my no meansexhaustive, belongs to the group that comprises fruit juices, milk,soy-based beverages, rise-based beverages, energy drinks, etc.

Yet another embodiment of the polyphenolic composition obtained fromunfermented malt and hops provided by this invention is related to itsuse in dietary supplements and/or a generic foodstuff.

The relative quantity ratios in Table 1 are also important in the lightof the ‘synergistic’ type biological activity that can be attributed tothe polyphenols that make up the polyphenolic composition provided bythis invention and its embodiment as a functional beverage.

The polyphenolic profile of the functional beverage in question ischaracteristic and original above all if compared to that ofnon-alcoholic beers.

The functional beverage has an intermediate profile between a dark and alight beer and this profile is characterized by the presence ofchlorogenic acid. Chlorogenic acid, an ester of caffeic acid, rarelypresent in the list of phenolic compositions identified in the mostcommon types of beer (light beer, dark beer, and non-alcoholic beer)instead appears to be present and in measurable quantities in the listof polyphenols of the functional beverage. Chlorogenic acid is known forits anti-oxidant power. The characteristic presence of chlorogenic acidin the polyphenolic composition obtained from malt and hops, canrepresent a specific marker of this composition. The pharmaceuticalindications of the composition are the following: treatment of chronicand degenerative pathologies (cardiovascular, neuro-degenerative,autoimmune, metabolic disorders and tumors). In the literature are knownpublications where the use of polyphenolic compositions is described asan aid in treating chronic and degenerative pathologies, like: FrancescoVisioli et al. (2011) and Daniele del Rio et al. (2013).

The polyphenolic composition according to the invention can beadministered as a food, as a dietary supplement, or as a pharmaceuticalto animal and elderly, adult, adolescent, children, and infant.

In a particular embodiment, the polyphenolic composition, according tothe present invention, is provided in liquid form, as a functionalnon-alcoholic beverage, using the method explained above or as a powderto be dissolved in a liquid, like water or milk or fruit juice. Thetotal quantity of natural polyphenols in the above-mentionedpolyphenolic composition in liquid form as a functional beverage, or asa non-alcoholic beer, is greater than 250 mg/l.

The following examples can be considered, by way of example and not oflimitation, of this invention.

Example 1

By way of example, for the production of 100 hl nominal of theabove-mentioned beverage, characterized by a final value of 12.87 PlatoDegrees, below are the quantities of raw materials: 1.8 kg of Pilsenbarley malt; 10 kg of toasted “dark” roasted barley malt; 4.0 kg of Hopsextract at 30%; 1.8 kg of Hops pellets 4.6%; 2.8 kg of Calcium Sulphateand 1.8 kg of phosphoric acid at 75%.

According to the method provided in this invention, the thermal cycle102 provides the processing of the A and B mixtures of malt and water(containing respectively 20% and 80% of the malt) at differenttemperatures and times according to the following table, where theprocess parameters are indicated:

Temperature Time Process details (° C.) (min) Infusion of the 20% maltmixture 60 17 Heating 66 1 Pause 66 20 Heating 100 16 Boiling 100 15Pause 100 5 Cooling 50 14 Addition of the 80% malt mixture 50 15 Pause50 20 Heating 63 9 Pause 63 40 Heating 72 6 Pause 72 40 Heating 76 5Pouring into the filtration tank 11

The filtration step 103 takes place during the preparation of the wortin the “cooking room” with the aim to remove the spent grains from thewort. This takes place in the filtration tank by the force of gravity;said filtration consists of a filtration of the wort and is carried outfor the purpose of separating the solid component (spent grains) fromthe liquid component (beer wort). In particular, the separated spentgrains are washed three times for the purpose of extracting morenutritional substances from the spent grains themselves.

Temperature Time Quantity Process details (° C.) (min) (hl) Pause for 5minutes in 76 5 90 the filtration tank Cloudy wort 76 7 First wort 76 8453 First washing 76 20 15 Second washing 76 25 15 Third washing 76 15 10

The boiling and hopping step 104 provides the boiling of the filteredwort and hopping. This process consists of the filtered wort beingcollected in a storage tank called “5th heater”. Once the 5th heater isfull, and so at the end of the wort filtration, the wort is sent in theWhirlpool where the successive heating and boiling phases will takeplace. The adding of the hops (4.0 kg of hops extract at 30%, 1.8 kg ofHops pellets 4.6%) takes place when the Whirlpool is full, so when allthe filtered wort has been poured from the 5th heater to the Whirlpooland heating can begin.

Temperature Time Quantity Process details (° C.) (min) (hl) 5^(th)heater full 76 144 99 (collection of the filtered wort from thebeginning of the first wort phase to the end or the third washing)Whirlpool full (pouring from 5^(th) 76 20 99 heater to the Whirlpool)Heating from 76 55 99 to 108 Boiling + pause 108  60 93 Stripper +cooling from 99 53 93 to 2

The phase denominated “stripper” consists of the injection of steam intothe wort, from below upwards, before the cooling, in order to removeunpleasant volatile compounds released with the boiling.

The wort cooling step 105 calls for the cooling of the wort until itreaches a temperature of 2° C. to send it into the aging tank and hencestart the storing stage 106. Cooling is obtained through a plate heatexchanger with cold water cross-flow.

The storage step 106 consists of the transfer of the wort into an agingtank for a storage time of about 3 days, without the addition of yeasts,contrary to what occurs during the beer production process where yeastsare added during this stage.

The filtration phase 107 consists of the use of Diatomaceous earth toremove the impurities from the aged beverage, thereby clarifying it. Thefiltration phase 107 takes place in a room called the “filtration room”using a horizontal pressure filter with cardboard filtering layers andusing Diatomaceous earth as a technological adjuvant. Generally, thesequestrants used during this phase also reduce the polyphenol content.Some beer producers use sequestrants to deliberately reduce or eliminatethe polyphenol content, as these compounds generate cloudiness.

In a further development of the invention, the filtration phase 107 isreplaced by a decantation phase 113, which consists of the removal ofimpurities from the aged beverage by collecting the upper part with noimpurities; the replacement of the filtration phase 107 with adecantation phase 113 results in a further increase of polyphenols inthe polyphenolic composition.

The treatment phase 108 consists of adding coloring malt extract andascorbic acid. The coloring malt extract is added to mask the cloudinessand reduce the degree of the polyphenols to a minimum, thereby boostingthe functional capacity of the functional beverage. It follows thebottling 109 and final pasteurization 110 phases. Further passages ofthis method may include drying 111 or lyophilization 112. In a preferredrealization the drying process 111 include also the 50° C. treatment.

The total quantity of polyphenols in the polyphenolic composition inliquid form as a functional beverage is about 300 mg/l, which iscomparable to the quantity of polyphenols in a typical light beer (>250mg/l) and almost double that of non-alcoholic beers on the market.

TABLE 2 comparison of total polyphenol concentrations PolyphenolsPolyphenols Type of beer (mg/l)¹ (mg/l)² Functional Beverage 300 — LightBeer 303 280 Dark Beer 533 420 Non-alcoholic Beer — 122 ¹The datacollected concerning the polyphenolic composition of the functionalbeverage were obtained at CNR laboratories. ²The data collectedconcerning the polyphenolic composition of the different beers wereobtained from the Phenol -Explorer data bank(http://phenol-explorer.eu/), which are the averages calculated usingmeasurements taken by various authors.

The polyphenolic composition in liquid form as a functional beverageshows a characteristic polyphenolic profile that stands, more or less,in an intermediate level between the polyphenolic profile of light beersand the polyphenolic profile of dark beers, and is also obviouslydifferent from the polyphenolic profile of non-alcoholic beers. Thepolyphenolic composition of the functional beverage provided with thisinvention is characteristic and new compared to standard polyphenoliccompositions of beers in that it contains measurable quantities ofchlorogenic acid, generally not reported in the literature as acomponent of the polyphenolic compositions of beers.

By way of example, Table 3 illustrates the polyphenolic profile of thepolyphenolic composition in functional beverage form obtained with themethod described according to this invention, with the detail of therelative ratios between the polyphenolic molecules present.

TABLE 3 polyphenolic profile of the polyphenolic composition infunctional beverage form Relative ratios of the phenolic Main phenoliccompounds of the compositions (Caffeic Acid molecule polyphenoliccomposition concentration is set as equal to 1) Caffeic Acid 1Chlorogenic Acid 0.92 m-Coumaric Acid 1.15 p-Hydroxybenzoic Acid 1.26Sinapinic Acid 1.48 Epicatechin 1.85 Protocatechuic Acid 1.92 Catechin2.66 p-Coumaric Acid 5.70 Ferulic Acid 9.40 Vanillic acid 2.74 ¹ Thedata collected concerning the polyphenolic composition of the functionalbeverage were obtained at CNR laboratories.

TABLE 4 Comparison of functional beverage polyphenolic profile vs. darkbeer, light beer, and non-alcoholic beer. Non- Functional Dark Lightalcoholic Phenolic compound beverage¹ beer² beer² beer² Caffeic Acid 1 11 1 Chlorogenic Acid 0.92 0 0 0 m-Coumaric Acid 1.15 0 0.66 0p-Hydroxybenzoic Acid 1.26 2.33 32 0.73 Sinapinic Acid 1.48 1 0.66 0.73Epicatechin 1.85 0.33 2 0.55 Protocatechuic Acid 1.92 1.33 1.66 27Catechin 2.66 0.67 3.66 10 p-Coumaric Acid 5.70 1.67 3.33 4 Ferulic Acid9.40 3 8.66 12 Vanillic acid 2.74 5.67 2.33 3 ¹Experimental data of thepolyphenolic composition in functional beverage form obtained from CNRlaboratories. ²The data collected concerning the polyphenoliccomposition of the different beers were obtained from the Phenol-Explorer data bank, which are the averages calculated using

Example 2

The polyphenolic composition obtained with the method describedaccording to this invention has demonstrated an anti-tumor activity inmurine models at a dosage D between 3 and 60 mg/kg, or higher than 3 andlower than 60 mg/kg, or 3<D<60.

In particular, the anti-tumor activity, both in the onset phase of thecancer and in the reduction of existing tumor, is noticeable at doses of3 mg/kg; with higher doses of 30 mg/kg, the polyphenolic compositionacts mostly by preventing the transformation of preneoplastic lesions intumors.

The anti-oxidant activity is demonstrable at doses of 30 mg/kg; asignificant reduction in the peroxides in the serum can be noticed withdoses of 100-300 mg/kg.

TABLE 5 correspondence between dosages and volumes of the polyphenoliccomposition administered to murine models and humans. Concentration ofConcentration of the polyphenolic the polyphenolic compositioncomposition by weight by volume Mouse Man 1 g/kg 8.5 ml/kg 0.3 ml 600 ml30 mg/kg 0.25 ml/kg 0.008 ml 17.5 ml 3 mg/kg 0.025 ml/kg 0.0008 ml 1.75ml

Table 5 illustrates a series of equivalences to calculate thecorrespondence in volume of polyphenolic composition, administered inbeverage form, administered to a man of an average weight of 70 kg toobtain the desired biological effects as observed in mice, which is tosay anti-tumor and anti-oxidant actions at indicated doses of 3 and 30mg/kg. As can be seen in the table above, by extrapolating the dataobtained on mice to man, the quantity of the polyphenolic composition,administered in functional beverage form, administered to a subject withan average weight of 70 kg are extremely low; this indicates anextremely active composition at very low doses, as is demonstrated bythe experimentations that follow.

The anti-tumor activity was studied in vitro on pre-clinical models(cellular lines and animal models) using the above-mentioned functionalcomposition in lyophilized form and/or dried. The studies conductedhighlighted the following effects:

1. Reduction in the vitality of cell lines of various origins; Reductionof the vitality of HL-60 cell lines. These cells were selected in thatthey were previously used for a screening of the antiproliferativeactivity of 48 commercial beers (Tedesco et al., 2005). The HL-60 derivefrom a human acute promyelocytic leukemia and when treated for 24 h with10% (v/v) of functional beverage, corresponding to about 6 mg of dryweight and about 60 μM equivalent of quercetin, in terms of totalpolyphenols, demonstrated a reduction in vitality of about 40% (neutralred spot assay). Tedesco et al., 2005, reported that this cytotoxicityis to be attributed, bona fide, to the activation of cellular death byapoptosis. Finally, it is useful to report that in lymphocytes fromperipheral blood prepared by normal subjects and used as a “control ofnormal cells” compared to HL-60, the functional beverage, under the sameexperimental conditions, induced a cytotoxicity of less than 5%,indicating greater effectiveness against the malign phenotype.

2. Reduction of the formation of aberrant crypts, foci, polyps andtumors induced by the AOM in an experimental model of colorectal tumorsin mice, with oral administration of the polyphenolic composition.

The anti-oxidant activity was demonstrated by measuring peroxideconcentration (equivalents of H2O2) in the serum of treated mice; theconcentration of peroxides is an index of the oxidative stress status ofthe organism. The experimentation included the use of the polyphenoliccomposition in the form of samples concentrated by drying. Asillustrated in Table 5, treatment by drying causes a lesser reduction inpolyphenols than lyophilization. Moreover, a slight increase inanti-oxidant activity and cytotoxicity of the dried samples was evident.

TABLE 6 content in polyphenols, anti-oxidant activity and cytotoxicityof the lyophilized and dried samples of polyphenolic composition.Anti-oxidant Polyphenolic Polyphenols activity Vitality composition (μMEqQ) (DPPH %) (% Ctrl) liquid (control) 887 9.2 65 Lyophilized 558 7.1265 Dried 748 10.9 60

In the following is showed experimental details of the tests conductedon polyphenolic composition samples according to this invention.

Experiment 1

The first experiment consisted in the administration of the polyphenoliccomposition in dried form (hereinafter db0%) in mice treated withazomethane (AOM); the experimentation lasted 14 weeks.

The treatment with AOM called for the administration of 10 mg/kg viaintraperitoneal injection in 4 successive doses over 4 consecutive weeks(one dose every weekend). The dried db0% was administered orally 5 timesper week for each week, beginning with the first week to the fourteenth,following which the mice were sacrificed.

The controls were represented by mice that were inoculated viaintraperitoneal injection 5 ml/kg of physiological solution (AOMcontrol) and mice administered spring water (db0% control).

The dosage used for the test was a range of doses between 3-300 mg/kg.

This dosage was identified by conducting a preliminary acute toxicitytest, in which it was found that at doses between 5-10 g/kg, the driedproduct caused the death of animals; on the other hand, doses between0.3 and 1 g/kg resulted in a reduction in the body weight of the mice,which became significant at doses of 1 g/kg. The maximum dose used wastherefore 300 mg/kg.

Following the treatment with AOM, it was demonstrated that, over aperiod of 13 weeks, the mice developed both aberrant crypt foci(preneoplastic lesions); in the colon-rectum of the mice there were alsonoticeable polyps and tumor.

FIGS. 2A and 2B illustrate the effect of administering differentconcentrations of the dried db0% (3, 30, 100, 300 mg/kg) on theformation of total aberrant crypt foci (FIG. 2A) and foci consisting of4 or more aberrant crypts in the colons (FIG. 2B) of the mice treatedwith AOM.

FIG. 3 illustrates the effect of administering different concentrationsof the dried db0% (3-30-100-300 mg/kg) on the formation of polyps (FIG.3A) and tumors (FIG. 3B) induced in the colons of mice treated with AOM.

The results indicate the anti-tumor activity of the dried db0% oncolorectal disease. In particular, it highlights that the extract of thelowest dosage, or 3 mg/kg act as soon as the first phase of the onset ofthe tumor, or by reducing the formation of the preneoplastic lesionsduring the transformation of the preneoplastic lesion in neoplasticlesions (polyps and tumors).

The dosage of 30 mg/kg acts mainly during the second phase or, in otherwords, by impeding the transformation of preneoplastic lesions inneoplastic lesions (polyps and tumors), especially in comparison toprevious dosages.

In general, a lesser efficiency of the dried product at highconcentrations was noticed when compared to low concentrations.

This behavior can be observed in many experiments where rawextracts/preparations containing bioactive molecules of differentnatures are used and can be the result of the combination of variousevents that occur simultaneously (synergistic effect).

Some of these events may depend on different bio-availability and/orbio-transformation of the bioactive compounds, on hormetic phenomenonand/or induced adaptation, or on the synergistic/additive effects ofthese compounds.

Experiment 2

The second experiment consisted of the measuring of the concentration ofperoxides in the serum of mice treated with AOM compared to untreatedcontrols and the verification of the protective effect of the db0%.

The concentration of peroxides (measured as equivalents of H2O2) is aparameter that is an index of the oxidative stress of the individual.

FIG. 4 highlights the concentration of peroxides in samples of serumfrom treated mice with different concentrations of dried db0%(3-30-100-300 mg/kg) both in the presence and absence of AOM.

FIG. 4 illustrates how the treatment with AOM to which the mice weresubjected, causes a significant increase in the concentration of theperoxides in the serum; the treatment with db0% causes a reduction inthe concentration of peroxides in the serum of mice and, in particular,at higher concentrations of 100-300 mg/kg, the concentration ofperoxides become less than the control value.

Even in the last column, the administering of db0% at a concentration of300 mg/kg in animals not treated with AOM caused a reduction in theconcentration of peroxides in the serum, confirming the anti-oxidantpower of the db0% preparation.

The advantages of this invention are represented by having perfected aninnovative method that preserves the polyphenolic content in barley maltnaturally, without having to add polyphenols, beginning with rawmaterials that are easy to find. One of the advantages of this inventionis represented by the doses necessary to achieve a potentiallytherapeutic action; indeed, in the experiments reported, it is thelowest dosage, or 3 mg/kg, that causes the most important therapeuticactivity in both preneoplastic lesions and tumoral lesions; in making acorrespondence and calculating the quantity in ml that a man of anaverage weight of 70 kg should take to obtain the anti-tumor effectobserved with concentrations of 3-30 mg/kg, extremely low values ofabout 2-20 ml were obtained. As a result of the above, this inventiondescribes a method for the production of a polyphenolic composition fromunfermented malt and hops, wherein the illustrated embodiments provide acomposition with a high content of natural polyphenols and ananti-oxidant and anti-tumor action in experimental models at doses 100times lower than toxic ones.

The object of the invention is susceptible to numerous modifications andvariants, all under the same inventive concept disclosed in the attachedclaims.

All parts may be replaced with other technically equivalent elements,and the materials may differ according to needs, without departing fromthe scope of protection of the present invention.

Although the object was described with particular reference to theattached figures, the reference numbers used in the description and inthe claims are used for a better understanding of the invention and donot constitute any limitation to the disclosed scope of protection.

BIBLIOGRAPHY CITED

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1. A polyphenolic composition comprising caffeic acid, chlorogenic acid,m-coumaric acid, p-hydroxybenzoic acid, sinapinic acid, epicatechin,protocatechuic acid, catechin, p-coumaric acid, ferulic acid, andvanillic acid.
 2. The polyphenolic composition of claim 1, wherein thecomposition has the following weight ratios between polyphenoliccomponents compared to caffeic acid set equal to 1: caffeic acid 1chlorogenic acid 0.83-1.01 m-coumaric acid 1.04-1.27 p-hydroxybenzoicacid 1.13-1.39 sinapinic acid 1.33-1.63 epicatechin 1.67-2.04protocatechuic acid 1.73-2.11 catechin 2.39-2.93 p-coumaric acid5.13-6.27 ferulic acid 8.46-10.3 vanillic acid 2.47-3.01
 3. Thepolyphenolic composition according to claim 1, wherein the compositionis in a liquid, powdered, dried, or lyophilized form.
 4. Thepolyphenolic composition according to claim 1, wherein the polyphenoliccomposition is for administration as food or beverage, as foodsupplement or drug, in solid form such as tablets, pills, hard capsules,powders, granules, suppository medicines, in semi-solid form such asgel, ointments, lubricants, pastes, in liquid form such as beverages,syrups, vials, drops, eye drops.
 5. The polyphenolic compositionaccording to claim 1, wherein the polyphenolic composition in is inliquid form, as a non-alcoholic functional drink, or powdered to bereconstituted in a liquid medium.
 6. A method of treating a chronic anddegenerative disease in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of thepolyphenolic composition of claim 25, wherein the chronic anddegenerative disease comprises at least one selected from the groupconsisting of cardiovascular, neurodegenerative, autoimmune, metabolicand tumoral diseases.
 7. The method according to claim 6, wherein thesubject is an animal or a human.
 8. A beverage or beer not obtained byfermentative methods, comprising the polyphenolic composition of claim25 and is free of ethyl alcohol and having a concentration of naturalpolyphenols greater than 250 mg/l.